Cell analysis apparatus, including flow cytometers and microfluidic analysers, are conventionally used to analyze and, at times, sort cells. Whether in order to sort X-chromosome sperm cells from Y-chromosome sperm cells, or to accomplish some other particle analysis (e.g., cell analysis), flow cytometers in particular, have an established reputation as an effective, albeit imperfect, analysis apparatus. Apparatus featuring microfluidic technologies, while representing a promising approach to the difficult problem of rapid particle analysis, have a less established reputation and are currently the subject of much attention and innovative design effort; accordingly, they are not used commercially to the extent the “tried-and-true” flow cytometers are.
Nonetheless, as mentioned, those using flow cytometers would welcome improvements, especially those that increase the proportion of cells whose analysis results are reliable. For example, the analysis results of 25% to 40% of sperm cells that the conventionally best flow cytometers analyze are unreliable; such cells typically go to waste. Of course, particularly in applications where the analyzed sperm cells are particularly valuable (sperm from a prize bull, an endangered species, as but two examples) such waste is highly undesirable. Further, such waste comes with wasted apparatus use time, and may discourage owners of, e.g., prize bulls, from selling their bull semen for sorting. Such problems, in general, are not unique to sperm sorting applications; indeed, any application that seeks to properly analyze (i.e., so as to produce reliable results) aspherical cells may find that conventional flow cytometers result in 25% to 40% of wasted cells.
The specific problem with flow cytometers, and perhaps certain microfluidic apparatus, stems from the difficulty in fully orienting (in plane that is orthogonal to the flow) cells, as many flow cytometer designs, in order to properly analyze aspherical cells (including but not limited to sperm cells) require not only that a cell be oriented relative to a flow axis (such that the cell long axis is parallel to the flow axis, which is relatively easy to do to all, if not 98+% of cells passing through the channel), but also that the cell be properly radially oriented, such that the long axis of the flow orthogonal, cell cross section (typically a non-circular cross-section) is aligned with an intended, flow orthogonal, cell cross section long axis alignment line that is defined by the channel. The reason for the need for such radial orientation has to do with the fact that the most reliable electromagnetic (EMR) detector readings (e.g., readings of EMR intensity emitted as a result of the cell illumination): (1) are of EMR (electromagnetic radiation) emitted out a lateral side (as opposed to the edge) of the cell/cell portion; and (2) result when the cell is illuminated upon projection of EMR on a lateral side. Such reliable readings can then be compared to yield accurate conclusions about an intrinsic characteristic of the cell (e.g., whether a sperm cell bears an X or Y chromosome).
In order to achieve such reliable readings, certain known flow cytometer designs employ a radially orienting channel (including a radially orienting nozzle tip and/or a beveled injection needle, as examples) designed to radially orient a cell such that a fixed EMR projector projects EMR at a lateral side of the cell (e.g. the lateral side of a flow orthogonal cross section of a sperm cell's head) and EMR emitted from a lateral side of the cell as a result of such illumination (e.g., as a result of fluorescence by stained DNA that are illuminated) can be read by a fixed detector. However, in order to determine whether a cell is in a fully radially oriented position, a different EMR detector is positioned to detect EMR emitted from the edge of the fully oriented cell (e.g., an edge of a flow orthogonal cross section of a sperm cell's head); readings from this “intended” “edge-on” detector are compared with the readings of the “intended” “side-on” detector. If indeed the reading from the intended “edge-on” detector (which may be said to provide information relative to said cell orientation) relates to the reading of the intended “side-on” detector in a manner that is found during a full radial orientation (for example, the reading from the intended “edge-on” detector is twice the reading of the intended “side-on” detector), then the intended radial orientation of such cell was in fact effected, the cell was illuminated properly (e.g., from a lateral side) and the reading from the detector established to detect EMR emitted out the lateral side of the cell (again, a lateral side of a flow orthogonal cell cross section) can be used to make a conclusion as to an intrinsic characteristic of the cell (e.g., whether the cell is X chromosome-bearing or Y chromosome-bearing).
Such conventional “two orthogonal detector” protocol relies on the well-known artifact effect where, e.g., the intensity of EMR emitted out the edge of a flow orthogonal cross-section of the head of a sperm cell as a result of a lateral side impinging illumination of such cell is twice as great as that of EMR emitted out the lateral side of a flow orthogonal cross-section of the head of such sperm cell as a result of such illumination. In general, the basic full radial orientation assurance protocol is an effective manner by to determine whether a detector reading is reliable; it is, in fact, employed in aspects of the inventive technology. However, the conventional EMR projector and detector configuration—while adequate to determine when a cell is fully radially oriented—does not address the problem of how to generate reliable readings from cells that are not fully radially oriented. Aspects of the inventive technology disclosed herein seek to achieve reliable readings from cells that, using conventional systems, would go to waste (because, of course, their less than full radial orientation renders detector readings unreliable). As such, aspects of the inventive technology may reduce waste as compared with conventional technologies. Aspects of the inventive technology, particularly those that seek to increase the percentage of analyzed cells as to which reliable conclusions regarding an intrinsic cell characteristic (again, every cell that is illuminated and whose emitted EMR is detected, regardless of whether such cell is fully oriented is considered an analyzed cell), may enable retrofitting of conventional flow cytometers so as to increase such percentage.
Additional aspects of the inventive technology address cell illumination configurations in which at least one additional electromagnetic radiation projector is located downflow of a “most upflow”, or first EMR projector, where all such projectors (e.g., a reflector or an EMR source) are established to effect the cell illumination by projecting electromagnetic radiation at the cell. Such “axially spaced illumination” embodiments of the inventive technology, which, similarly to the “off-axis” detector technologies, may find particular application not only to analysis systems that seek to fully radially orient cells, but also to systems that do not seek to fully radially orient cells. Indeed, aspects of the inventive “axially spaced illumination” technology, particularly those in which EMR projectors define flow orthogonal projector axes that together define a non-zero angle (i.e., where the axes, again, each in an axially separated flow orthogonal plane, when overlayed, define a non-zero angle) may be able to reduce the percentage of cells that are wasted in radially orienting systems; where a sufficient number of such axially spaced EMR projectors are so established, acceptable percentages of cells as to which reliable emitted EMR detection results cells may be obtained even in non-orienting systems.
As such, at least one embodiment of the inventive technology seeks to reduce the percentage of cells that are wasted in radially orienting analysis systems.
At least one embodiment of the inventive technology seeks to reduce the percentage of cells that are wasted in analysis systems that do not seek to radially orient cells.
At least one embodiment of the inventive technology seeks to enable retrofitting of existing radially orienting systems so as to reduce the percentage of cells that are wasted, perhaps by 10% to 20%.
A goal of at least one embodiment of the inventive technology is to provide an analysis system that obtains reliable emitted EMR detector readings from cells from that prior art systems are unable to reliably detect.
A goal of at least one embodiment of the inventive technology is to provide a multiple illumination system configured so as to illuminate cells from various angles, thereby either: obtaining reliable emitted EMR readings from cells that, in radially orienting systems, would otherwise proceed to waste; or obtaining acceptable percentages of analyzed cells having reliable EMR readings in analysis systems that do not seek to radially oriented cells.
A goal of at least one embodiment of the inventive technology is to provide a multiple illumination system that illuminates cells only to the extent necessary.
A goal of at least one embodiment of the inventive technology is to obtain reliable detector readings—readings that can be used to yield accurate information relative to an intrinsic cell characteristic—from a cell whose radial orientation is from 10 to 45 degrees away from full radial orientation.
Of course, other goals and advantages of the inventive technology are revealed in the disclosure provided herein, whether explicitly or implicitly.